The products of the MYC family of protooncogenes, including c-Myc, N-Myc, and L-Myc proteins, function in cell proliferation, differentiation, and neoplastic disease (1; see the appended Citations). However, there is as yet no consensus as to the molecular mechanism by which Myc mediates its biological effects. The Myc proteins are nuclear phosphoproteins with short half-lives and nonspecific DNA-binding activities (2). Functionally important regions exist as both the amino and carboxyl termini of the c-Myc protein (3-5). Indeed, the carboxyl-terminal 85 amino acids of the Myc family proteins share significant sequence similarity with two classes of transcription factors, the basic region helix-loop-helix (bHLH) and basic region leucine zipper (bZip) proteins, both of which have basic regions adjacent to their dimerization domains. The bHLH family includes over 60 proteins in vertebrates, yeast, plants, and insects; many, if not all, exhibit nuclear localization, are sequence-specific DNA-binding proteins, and function as transcriptional regulators(6). The region of sequence similarity shared to Myc and other proteins in this class is a critical determinant of function and contains a stretch of basic amino acids followed by two putative amphipathic a helices that flank an W-type loop (7,8). Studies of several other bHLH proteins have demonstrated that the HLH region mediates formation of homo- or heterodimers, which in turn permits the basic regions to form a DNA contact surface (9-11). Myc family proteins differ from the bHLH family in that adjacent and carboxyl-terminal to their bHLH motif is another a helix that contains a heptad repeat of leucine residues. This structure is characteristic of the dimerization domains of the bZip family of transcriptional regulators (12). The array of nonpolar amino acids forms a hydrophobic face long the amphipathic helic, facilitating specific association of bZip proteins through a parallel coiled-coil interaction (13). Dimerization is critical for DNA binding (14, 15).
For c-Myc there is substantial evidence that the bHLH region and the adjacent leucine zipper motif are functionally important. Deletions within these regions result in loss of alteration of transforming activity (3, 16) as well as reduction of the capacity of autoregulate endogenous myc expression and to inhibit cell differentiation (4, 5). In addition, a bacterially expressed fusion protein that contains the bHLH-Zip domains of c-Myc has sequence-specific DNA-binding activity (17).
It is also of interest to consider the myc oncogene in the context of tumor suppressor genes since, at least on theoretical grounds, it is precisely the proliferation-inducing effects of myc that one would expect to be opposed by genes of the tumor suppressor class. The notion that myc oncogene function is linked to cell proliferation is now supported by multiple lines of evidence. Much of this evidence has been summarized in recent reviews (18,19) and will be briefly reiterated here. First, c-myc expression is strongly correlated with cell growth. During exponential growth of many different cell types, c-myc-encoded mRNA and protein synthesis is maintained at a constant level throughout the cell cycle (20,21). By contrast, c-myc expression is essentially undetectable in quiescent (G.sub.0) cells and in most, but not all, terminally differentiated cell types. The down-regulation of c-myc expression during differentiation is likely to be a critical event since forced expression of exogenous c-myc blocks the induced differentiation of erythroleukemia cells and adipocytes (22, 23) while anti-sense inhibition of c-myc expression in HL60 cells leads directly to differentiation (24).
On the other hand, the entry into the cell cycle of quiescent cells is invariably accompanied by a large transient burst of c-myc expression within hours of mitogenic stimulation of both hematopoietic and nonhematopoietic cell types (25). Indeed, c-myc is prototypical of the class of immediate early response genes encoding labile mRNAs which can be induced (or superinduced) in the presence of protein synthesis inhibitors. That myc expression is important for entry into the cell cycle is suggested by experiments utilizing c-myc anti-sense oligonucletides, which appear to block the entry of mitogenically stimulated human T cells into S phase but not into the G.sub.1 phase of the cell cycle (26). Recent experiments using an artificially "activatable" c-myc-encoded protein (c-Myc) have demonstrated that quiescent fibroblasts can be made to enter the cell cycle following activation of c-Myc. Amazingly, this occurs in the absence of the induction of the other major early response genes, including jun and fos (27). Thus, c-myc expression may be sufficient for entry of G.sub.0 cells into the cell cycle.
Further support for the idea that Myc function is strongly linked to cell proliferation and differentiation comes from the vast amount of data demonstrating an association between the deregulation of myc family gene expression and neoplasia (for reviews, see 28-30). Oncogenic activation of myc by retroviral capture, promoter/enhancer insertion, gene amplification, and chromosomal translocations all appear to lead to abnormal and uncontrolled proliferation of numerous cell types. While these events frequency result in myc overexpression, they also result in a loss of the normal regulatory elements that control normal myc expression. A great deal of work has demonstrated that myc expression is normally regulated at multiple levels (for recent review, see 31), and it is the loss of such regulation which is believed to result in uncontrolled cell proliferation and a reduced capacity for terminal differentiation.
Although it is indisputable that Myc is involved in cell proliferation, it is less clear whether the functions of tumor suppressor genes, which are often through to act as negative growth regulators (see 32 for review), actually impinge directly on Myc function. One potential example of interaction between myc function and tumor suppressor gene activity has come from studies demonstrating that treatment of an epithelial cell line with TGF-.beta. results in transcriptional repression of c-myc which is reversible by agents (adenovirus E1A, SV40 R antigen) that sequester the Rb gene product (33,34). While these data do not necessarily indicate a direct interaction between Myc and Rb they at least hint at the possibility that the functional pathways of these two gene products may be intertwined. In addition, it is possible that Myc might interact directly with an as yet uncharacterized tumor suppressor protein. It is clear that more details concerning the molecular mechanism of Myc function are required in order to explore more fully the possibility of direct interaction between Myc and tumor suppressor gene products. One approach is to define the interactions of Myc protein with other cellular proteins, as well as with nucleic acids. Such studies may help to elucidate Myc's molecular function and reveal the circuitry through which proliferation suppression factors may interact with Myc.
The biological importance of and structural similarities in the carboxyl terminus of c-Myc suggest that Myc functions as a component of an oligomeric complex. While Myc self-association has been demonstrated with relatively high concentrations of bacterially expressed Myc protein (35), coprecipitation, chemical crosslinking, and dimerization motif chimeras fail to demonstrate homodimerization of Myc under physiological conditions (1, 36, 37). Because functionally relevant interactions occur among members of the bHLH and bZip classes (9, 15, 38, 39), and c-Myc has not yet been found to associate with members of either group (10, 15, 16), we hypothesized that Myc function may depend on heterotypic interaction with an unknown protein. We now describe the cloning of such a Myc binding factor, termed Max, and its regulatory factor, termed Mad.